| 摘要: |
| 目的:构建携带人巨细胞病毒(hCMV) UL144基因的重组腺病毒,研究UL144基因修饰的树突状细胞(dendritic cell,DC)的功能变化。方法:从hCMV-DNA阳性患者外周血提取cDNA,采用PCR技术扩增出UL144基因。采用AdEasy系统构建携带hCMV UL144基因的重组腺病毒载体pAd-UL144,转染HEK293细胞后包装产生重组腺病毒Ad-UL144。通过RT-PCR检测目的基因的表达。将此重组腺病毒转染小鼠骨髓来源的DC,采用流式细胞术检测DC表面分子,采用ELISA技术分析DC培养上清中的细胞因子,采用3H-TdR掺入法检测UL144基因修饰的DC激活T细胞增殖的能力。结果:成功将UL144基因片段克隆至载体上,并经HEK293细胞包装出病毒颗粒,经测定病毒滴度为3×1010 pfu/ml。流式细胞术检测显示,UL144基因修饰的DC表面分子CD80、CD86和I-Ad表达水平低于对照组(P<0.01)。ELISA分析表明,UL144基因修饰的DC分泌TNF-α、IL-6和IL-1β的能力减弱(P<0.05)。UL144基因修饰的DC介导的T细胞增殖功能明显低于DC对照组(P<0.01)。结论:UL144基因修饰的DC具有维持相对未成熟状态的能力,并减弱了对抗原特异性T效应细胞的刺激功能。 |
| 关键词: 人巨细胞病毒 UL144基因 疱疹病毒侵入介体 树突状细胞 免疫耐受 |
| DOI：10.3724/SP.J.1008.2009.0874 |
| 投稿时间：2009-01-21修订日期：2009-06-04 |
| 基金项目:国家自然科学基金(30600244). |
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| Clone and expression of human cytomegalovirus UL144 gene and its effects on dendritic cells |
| WANG Huai-zhou,CAI Gang,SHEN Qian* |
| (Department of Clinical Diagnosis,Changhai Hospital,Second Military Medical University,Shanghai 200433,China) |
| Abstract: |
| Objective:To construct a recombinant adenovirus vector carrying human cytomegalovirus (hCMV) UL144 gene and to explore the biological characteristics of UL144 gene-modified DCs.Methods: The UL144 gene was amplified from hCMV DNA,which was extracted from hCMV-DNA positive serum.The recombinant adenovirus vector carrying hCMV UL144 gene was constructed with AdEasy system and then transfected into HEK293 cells to create recombinant adenovirus Ad-UL144.The expression of inserted gene was identified by RT-PCR.The recombinant adenovirus was then transfected into mice myeloid dendritic cells.The surface proteins of dendritic cells were analyzed by FACS,and cytokines in supernatant were detected by ELISA.T cell proliferation stimulated by gene-modified DC was examined by 3H-TdR uptake assay.Results: The UL144 gene was successfully cloned into the pAdEasy-1 plasmid.The recombinant adenovirus Ad-UL144 was packed in HEK293 cells,with a viral titer of 3×1010 pfu/ml.DCs infected with AdCMV-UL144 had markedly decreased surface expression of CD80,CD86 and I-Ad (P<0.01).The contents of TNF-α,IL-6 and IL-1β were significantly decreased in the supernatant of AdCMV-UL144 modified DCs(P<0.05).T cell proliferation ability induced by gene-modified DC was obviously lower than in the DC control group (P<0.01).Conclusion: UL144-modified DCs can maintain a relative immature status,and have reduced stimulating activity upon the proliferation and activation of T cells in vitro. |
| Key words: human cytomegalovirus UL144 gene herpes virus entry mediator dendritic cell immune tolerance |
